Arthropod-borne viruses (arboviruses) exist in a complex cycle between their arthropod vector and vertebrate host. West Nile virus (WNV), an arbovirus that causes significant human and veterinary disease, is maintained in nature by cycling between birds and Culex spp. mosquitoes. The dynamic mosquito-virus vertebrate host interaction is involved not only in the efficiency of enzootic transmission, but also in the pathogenesis of disease. Currently, the impact of mosquito transmission on WNV infection in the vertebrate is poorly defined. The objective of this research proposal is to further our understanding of the interaction of WNV with its vertebrate and invertebrate hosts in order to develop novel control and treatment strategies. Based on preliminary data that early viremia is 100-fold higher in chickens infected with WNV by mosquitoes compared to needle inoculation, the central hypothesis is that mosquito transmission enhances the earliest steps of WNV infection in its vertebrate host. Studies to test this hypothesis will use the following approaches: 1) viral replication in primary and secondary sites of replication will be compared for natural mosquito transmission and needle inoculation of WNV in chickens and mice; 2) different aspects of mosquito transmission of WNV will be tested for their effect on eady viremia in chickens, including viral dose, saliva, mosquito probing and mosquito-derived virus; 3) the mechanism responsible for the early viremia will be identified, and in particular the effect on the host's Type I interferon response will be examined in vivo and in vitro. Completion of this study will further elucidate the interaction of WNV and the mosquito with amplifying (bird) and incidental (mouse) hosts and will define the critical role that mosquito saliva plays in WNV transmission and disease. The research proposed in this application is significant because understanding this complex interaction is essential to developing innovative treatment and control strategies for WNV.